Composition and methods for assessing sensitivity and specificity of antibody detection reagents

ABSTRACT

Compositions and methods which are useful for determining the sensitivity and specificity of antibody detection reagents are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application based onInternational Application No. PCT/US2016/053311, filed Sep. 23, 2016,which claims the benefit of priority to U.S. Provisional PatentApplication No. 62/232,310, filed Sep. 24, 2015 and 62/368,069 filedJul. 28, 2016, each of which is incorporated herein by reference in itsentirety as if fully set forth herein.

STATEMENT REGARDING SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 13, 2018, isnamed B212-0008US_ST25.txt and is 85 KB in size.

FIELD

The invention relates to compositions and methods useful for determiningthe sensitivity and specificity of antibody detection reagents.

INTRODUCTION

Humans naturally carry ABO antigens and express anti-A and/or anti-B ifthey do not express A or B, respectively. In addition to ABO, there arein excess of 340 RBC alloantigens in humans, each of which vary personto person. While anti-A and anti-B develop in essentially all people as“naturally occurring” alloantibodies, antibodies to the otheralloantigens typically have to be induced by transfusion and/orpregnancy. However, only some patients make such antibodies. Once ananti-RBC alloantibody is made, then a patient often cannot be transfusedwith RBCs expressing the recognized alloantigen. It is for this reasonthat patients, prior to transfusion, are screened for the presence ofRBC alloantibodies.

SUMMARY

In one aspect, disclosed herein is a method to determine the specificityand sensitivity of an anti-human globulin including the steps of: a)binding the anti-human globulin antibody to be tested to a panel ofhuman antibodies of different subtype, and b) detecting the binding ofthe anti-human globulin to a subtype, thus determining the specificityand sensitivity of the anti-human globulin.

In some embodiments, the human antibody is an IgG. In some embodiments,the subtype includes IgG1, IgG2, IgG3, and IgG4. In some embodiments,the panel of human antibodies of different subtype includes the antigenbinding sites of PUMA1. In some embodiments, the panel of humanantibodies is at least one of PUMA 1 IgG1, IgG2, IgG3, and IgG4. In someembodiments, the human antibody is IgA, IgM, IgE, or IgD.

In other embodiments, the human antibody binds a member of the Kellblood group antigen system, for example, KEL1, KEL2, KEL3, KEL4, KEL5,KEL6, or KEL7. In particular embodiments, the Kell blood group antigenis K, Kp^(b), or Js^(b).

In further embodiments, the human antibody includes a heavy chainincluding at least one CDR selected from the group of CDR sequencesshown in FIG. 1.

In yet further embodiments, the human antibody includes a light chainincluding at least one CDR selected from the group of CDR sequencesshown in FIG. 2.

In other embodiments, the human antibody includes a heavy chainincluding one, two, or three CDR(s) selected from the group of CDRsequences shown in FIG. 1.

In other embodiments, the human antibody includes a light chainincluding one, two, or three CDR(s) selected from the group of CDRsequences shown in FIG. 2.

In other embodiments, the human antibody includes a heavy chainincluding at least a portion of the sequence shown in FIG. 1.

In other embodiments, the human antibody includes a light chainincluding at least a portion of the sequence shown in FIG. 2.

In some embodiments, the antibody or fragment thereof binds to anantigen with an affinity constant (K_(D)) of less than 1×10⁻⁸ M.

In some embodiments, the antibody or fragment thereof binds to anantigen with an affinity constant (K_(D)) of less than 1×10⁻⁹ M.

In some embodiments, disclosed herein is an expression vector includinga nucleic acid encoding the human antibody disclosed above.

In some embodiments, the expression vector is in a host cell, which caninclude a bacterial cell or a eukaryotic cell, such as a mammalian cell.

In some embodiments, disclosed herein is a human antibody which includesa heavy chain including at least one CDR selected from the group of CDRsequences shown in FIGS. 1, 3, and 5.

In some embodiments, disclosed herein is an antibody which includes alight chain including at least one CDR selected from the group of CDRsequences shown in FIGS. 2, 4, and 6.

In some embodiments, disclosed herein is a human antibody which includesa heavy chain including one, two, or three CDR(s) selected from thegroup of CDR sequences shown in FIGS. 1, 3, and 5.

In some embodiments, disclosed herein is a human antibody which includesa light chain including one, two, or three CDR(s) selected from thegroup of CDR sequences shown in FIGS. 2, 4, and 6.

In some embodiments, disclosed herein is a human antibody, whichincludes a heavy chain including the sequence shown in FIG. 1, FIG. 3,or FIG. 5.

In some embodiments, disclosed herein is a human antibody includes alight chain including the sequence shown in FIG. 2, FIG. 4, or FIG. 6.

In some embodiments, disclosed herein is a human antibody, whichincludes a heavy chain including the sequence of FIG. 1 and a lightchain sequence of FIG. 2.

In some embodiments, disclosed herein is a human antibody, whichincludes a heavy chain including the sequence of FIG. 3 and a lightchain sequence of FIG. 4.

In some embodiments, disclosed herein is a human antibody, whichincludes a heavy chain including the sequence of FIG. 5 and a lightchain sequence of FIG. 6.

In some embodiments, disclosed herein is an expression vector includingany one of the nucleic acids shown in FIGS. 1-6.

In some embodiments, the method is performed using a FACS assay, a geltesting assay, a tube testing assay, or a solid phase testing assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the heavy chain sequence (SEQ ID NOs: 3, 4, and 5) ofmonoclonal antibodies Puma 1 and 2 directed to KEL1 (K). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: DYYMK, SEQ ID NO: 114; CDR2: DLNPNNGDTFYNQKFKG, SEQ ID NO: 115;CDR3: CAREAGSSFGSSCNYWG, SEQ ID NO: 116) of the heavy chain areunderlined.

FIG. 2 shows the the light chain sequence (SEQ ID NOs: 7, 8, and 9) ofmonoclonal antibodies Puma 1 and 2 directed to KEL1 (K). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: KASQTVSKDVA, SEQ ID NO: 117; CDR2: YASNRYT, SEQ ID NO: 118; CDR3:QQDYSS, SEQ ID NO: 119) of the light chain are underlined.

FIG. 3 shows the heavy chain sequence (SEQ ID NOs: 10, 11, and 12) ofmonoclonal antibody Puma 3 directed to a common Kell epitope. Theshading indicates where the highly variable region begins. The CDRregions (CDR1: SYGVY, SEQ ID NO: 120; CDR2: IIWGDGSTNYQSVLRS, SEQ ID NO:121; CDR3: RGDYDVA, SEQ ID NO: 122) of the heavy chain are underlined.

FIG. 4 shows the light chain sequence (SEQ ID NOs: 14, 15, and 16) ofmonoclonal antibody Puma 3 directed to a common Kell epitope. Theshading indicates where the highly variable region begins. The CDRregions (CDR1: KASQTVSEVGTSLMH, SEQ ID NO: 123; CDR2: RTSNLEA, SEQ IDNO: 124; CDR3: QQS) of the light chain are underlined.

FIG. 5 shows the heavy chain sequence (SEQ ID NOs: 17, 18, and 19) ofmonoclonal antibody Puma 4 directed to KEL4 (Kp^(b)). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: NYWMN, SEQ ID NO: 125; CDR2: EIRLNSNNYATHYAESVKG, SEQ ID NO: 126;CDR3: NWDFAW, SEQ ID NO: 127) of the heavy chain are underlined.

FIG. 6 shows the light chain sequence (SEQ ID NOs: 20, 21, and 22) ofmonoclonal antibody Puma 4 directed to KEL4 (Kp^(b)). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: KASQDVSTVVA, SEQ ID NO: 128; CDR2: WASTRHT, SEQ ID NO: 129; CDR3:QQHYT, SEQ ID NO: 130) of the light chain are underlined.

FIG. 7 shows the specificity of monoclonal antibody Puma 1.

FIG. 8 shows the specificity of monoclonal antibody Puma 2.

FIG. 9 shows the specificity of monoclonal antibody Puma 3.

FIG. 10 shows the specificity of monoclonal antibody Puma 4.

FIG. 11 shows (A) the sequences of humanization of PUMA1 to human IgG1(SEQ ID NO: 23), IgG2 (SEQ ID NO: 24), IgG3 (SEQ ID NO: 25), and IgG4(SEQ ID NO: 26) and (B) the alignment of these sequences (SEQ ID NOs:23-26).

FIG. 12 shows recombinant generation of a humanized form of PUMA1 andits ability to bind to antigen positive RBCs, demonstrating amaintenance of binding after humanization of the IgG constant region.

FIG. 13 shows a general overview of how anti-human globulin (AHG) isused diagnostically in patients being screened for alloantibodies priorto transfusion.

FIG. 14 shows an overview of process to isolate and engineer RBC bindingantibodies (anti-Kell) that maintain the same antigen binding sites, butdiffer in their IgG subtype and allotype.

FIG. 15 shows the use of PUMA1 IgG1, IgG2, IgG3, and IgG4 to evaluatevarious anti-human globulin (AHG) preparations using a FACS assay.

FIG. 16 shows the use of PUMA1 IgG1, IgG2, IgG3, and IgG4 to evaluatevarious anti-human globulin (AHG) preparations using a gel testingassay.

FIGS. 17-22 show the use of PUMA1 IgG1, IgG2, IgG3, and IgG4 to evaluatevarious anti-human globulin (AHG) preparations using a tube testingassay.

FIGS. 23-25 show the use of PUMA1 IgG1, IgG2, IgG3, and IgG4 to evaluatevarious anti-human globulin (AHG) preparations using a solid phasetesting assay.

FIGS. 26A-26C illustrate specificity of PUMA1 for the K antigen andstrategy to generate PUMA1 variants. As shown in FIG. 26A, mouse RBCswere stained with PUMA1 antibody followed by secondary antibody(wild-type RBCs dotted line, K transgenic RBCs solid line). K transgenicRBCs were also stained with secondary antibody alone (dashed line). Asshown in FIG. 26B, human RBCs with a K+k+ phenotype were stained withPUMA 1 followed by secondary antibody (solid line) or with secondaryantibody alone (dashed line). RBCs with a K-k+ phenotype were stainedwith PUMA1 followed by secondary antibody (dotted line). FIG. 26Cprovides a diagram showing the general cloning and expression strategy.

FIG. 27 provides data obtained when K+k+ RBCs were incubated with eachof the indicated PUMA1 IgG isoallotype, followed by the test anti-IgGand relevant detection reagent (see methods) [dark(er) gray histograms].K-k+ RBCs were also incubated with each IgG isoallotype as a backgroundcontrol [light(er) gray histograms]. Isoallotypes that were notrecognized are indicated by (*).

FIGS. 28A, 28B provide data obtained when human K+k+ RBCs sere incubatedwith a titration of each human IgG subclass of PUMA1 in the indicatedamounts, followed by staining with the appropriate secondary antibodies(see methods). This was carried out for both of the indicated anti-IgGreagents, Ortho AHG in FIG. 28A, and Immucor Gamma AHG in FIG. 28B. Alldata are derived from mean fluorescent intensity determined by flowcytometry.

FIG. 29 provides amino acid basis for variation amongst IgGisoallotypes. For each IgG subtype, the *01 designation is the canonicalsequence. (SEQ ID NOs: 27-113).

DETAILED DESCRIPTION

Compositions and methods for determining the sensitivity and specificityof antibody detection reagents are provided herein.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such can, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges can independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges can be presented herein with numerical values beingpreceded by the term “about.” The term “about” is used herein to provideliteral support for the exact number that it precedes, as well as anumber that is near to or approximately the number that the termprecedes. In determining whether a number is near to or approximately aspecifically recited number, the near or approximating unrecited numbercan be a number which, in the context in which it is presented, providesthe substantial equivalent of the specifically recited number. The term“about” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, such as ±5%, such as ±1%, and such as ±0.1%from the specified value, as such variations are appropriate to performthe disclosed methods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided can be different from the actual publication dateswhich can need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

Additionally, certain embodiments of the disclosed devices and/orassociated methods can be represented by drawings which can be includedin this application. Embodiments of the devices and their specificspatial characteristics and/or abilities include those shown orsubstantially shown in the drawings or which are reasonably inferablefrom the drawings. Such characteristics include, for example, one ormore (e.g., one, two, three, four, five, six, seven, eight, nine, orten, etc.) of: symmetries about a plane (e.g., a cross-sectional plane)or axis (e.g., an axis of symmetry), edges, peripheries, surfaces,specific orientations (e.g., proximal; distal), and/or numbers (e.g.,three surfaces; four surfaces), or any combinations thereof. Suchspatial characteristics also include, for example, the lack (e.g.,specific absence of) one or more (e.g., one, two, three, four, five,six, seven, eight, nine, or ten, etc.) of: symmetries about a plane(e.g., a cross-sectional plane) or axis (e.g., an axis of symmetry),edges, peripheries, surfaces, specific orientations (e.g., proximal),and/or numbers (e.g., three surfaces), or any combinations thereof.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

The current disclosure provides for isolating a monoclonal anti-RBCalloantibody against a common human RBC antigen (Kell), and cloning outthe antigen binding domains of the antibody (both heavy and lightchain). The isolated antibody is called PUMA1. The antigen bindingdomains of the heavy chain were then cloned upstream and in frame withthe coding sequence for IgG1, IgG2, IgG3 and IgG4 human heavy chains.Expression vectors for these novel recombinant sequences wereco-transfected (along with the light chain) into CHO cells, followed bypurification of the recombinant antibodies (see FIG. 13). In this way, 4different antibodies (of the different IgG subtypes) were purified tohomogeneity and in the absence of IgGs of the other subtypes. Moreover,each PUMA1 IgG subtype binds to the same target epitope, allowing astandardization of affinities across each PUMA1 IgG subtype. Finally,since PUMA1 recognizes a common RBC alloantigen, this allows PUMA 1IgG1-IgG4 to serve as standards in each of the existing RBC antibodydetected platforms, so as to assess the ability of any given batch orpreparation of AHG (polyclonal or monoclonal) to bind each human IgGsubtypes and in different platforms.

Further refinement of this approach is the introduction of additionalsequence variations in the IgG constant regions. Humans have multipleknown variations in IgG constant regions (called allotypes if theyconstitute a new epitope). Additional variations have been describedthat are not known to generate epitopes, but nevertheless can change IgGstructure. It is unclear the extent to which any given AHG willrecognize any of these given sequence variants. By introducing thesevariants into the PUMA1 heavy chain vectors, a full panel of all knownIgG subtypes (IgG1-IgG4), and all known variants, provides novelreagents that can serve as quality control and characterizationdiagnostics for AHG in any existing platform that screens patients foranti-RBC alloantibodies. As the particulars of any given testingplatform can vary, AHG performances can vary.

Finally, this approach can be taken to make monoclonal PUMA1 of the IgM,IgA, IgE or IgD isotype, for standards to assess assays that usevariants of AHG to detect these other isotypes.

Additional application of the techniques described above and, forexample, with respect to isoallotypes below, extends to making novelantibodies (by the same general approach) so as to make reagents fortesting of diagnostics ability to detect antibodies to platelets, whiteblood cells, other tissues (auto, allo and xeno), viruses, bacteria,fungi, parasites, vaccines, and purified antigens. As such, the subjectmethods include identifying and/or manufacturing such regents.

The subject aspects also include methods of manufacturing or otherwiseproducing any of the subject systems, kits, assays or components, e.g.,reagents, thereof as well as methods of manufacturing or otherwiseproducing assays or components thereof which are operated according toany of the methods or method steps provided herein.

In the embodiments set forth herein, any one or more of thecharacteristics of the subject disclosure, e.g., methods, referring toIgG1, IgG2, IgG3, or IgG4 can also be applied in the same manner withrespect to any of the 29 IgG isoallotypes alone or in combination.Furthermore, any one or more of the characteristics of the subjectdisclosure, e.g., methods, referring to IgG1, IgG2, IgG3, or IgG4 canalso be applied in the same manner with respect to any subject, e.g.,mammal, e.g., human, immunoglobulin component. As such, the subjectdisclosure includes assays for assessing characteristics of suchcomponents, such as their presence or absence from a panel, as set forthherein.

Human immunoglobulin components which can be applied according to thesubject aspects include, for example, IgM, IgA1, IgA2, IgE or IgD, orany variants thereof. Human immunoglobulin components which can beapplied according to the subject aspects also include, for example,anti-human leukocyte antigen (HLA) antibodies or one or more isotypesthereof, such as HLA-specific immunoglobulin G antibodies.Immunoglobulin components which can also be applied include hyperimmuneimmunoglobulins from human immunodeficiency virus (HIV)-infected persons(HIVIG). Animal immunoglobulin components can also be utilized accordingto the subject embodiments.

In some embodiments, a subject, such as a subject from which one or moresamples or portions thereof, e.g., proteins are derived, is a “mammal”or a “mammalian” subject, where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, andrats), and primates (e.g., humans, chimpanzees, and monkeys). In someembodiments, the subject is a human. The term “humans” can include humansubjects of both genders and at any stage of development (e.g., fetal,neonates, infant, juvenile, adolescent, and adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe subject matter described herein can be applied in association with ahuman subject or one or more samples or aspects therefrom, it is to beunderstood that the subject matter can also be applied in associationwith other subjects, that is, on “non-human subjects.”

Isoallotypes

As designated above, IgG exists in 4 different subtypes, IgG1, IgG2,IgG3, and IgG4. There is genetic variation within IgG subtypes. Suchgenetic variants within IgG subtypes are referred to as isoallotypes.There are at least 29 different isoallotypes, with 7, 4, 15, and 3isoallotypes existing for IgG1, IgG2, IgG3, and IgG4, each respectively.The isoallotypes are referred to herein for each immunoglobulin bynumber and, in some cases, an additional designation such as “v2.” Alisting of the isoallotypes and their sequences is provided, forexample, in FIG. 29. In various embodiments, the reactivity of anti-IgGwith different isoallotypes is evaluated.

According to the subject embodiments, any one or more of thecharacteristics of the subject disclosure, e.g., methods, referring toIgG1, IgG2, IgG3, or IgG4 can also be applied in the same manner withrespect to any of the 29 isoallotypes alone or in combination.

In various embodiments of the subject disclosure, a monoclonal anti-Kantibody (PUMA1) was isolated, sequenced, and a panel of PUMA1 variantswas expressed including the 29 known IgG isoallotypes. The resultingpanel of antibodies was pre-incubated with K+ RBCs and was thensubjected to testing with currently approved anti-IgG, by flowcytometry, solid phase systems, gel card, and tube testing.

In some aspects of the disclosure, an FDA approved monoclonal anti-IgG(Gammaclone) failed to recognize 2 out of 15 IgG3 isoallotypes (IgG3-03and IgG3-13) and 3 out of 3 IgG4 isoallotypes (IgG4-01, 02, 03). Also,in some aspects of the subject disclosure, an FDA approved rabbitpolyclonal anti-IgG recognized each of the known human IgG isoallotypes.

In some aspects of the subject embodiments, methods are provided thatinclude, for example, determining the specificity and/or sensitivity ofan anti-human globulin. The methods can include, for example, binding ananti-human globulin antibody to a panel of human antibodies of differentsubtype. Aspects of the methods also can include detecting the bindingof the anti-human globulin to a subtype, such as IgG1, IgG2, IgG3, IgG4,or any one or more isoallotype of any of such immunoglobulins, and thusdetermining the specificity and sensitivity of an anti-human globulin.

The methods also include detecting and/or determining the absence ofbinding or coupling of the anti-human globulin to one or more subtype,such as IgG1, IgG2, IgG3, IgG4, or any one or more isoallotype of any ofsuch immunoglobulins, such as any one or combination of the isoallotypesprovided in FIG. 29. Such a method can be applied to identify “holes” inan assay where such an assay would not produce a useful result.

Various embodiments of the subject disclosure include detecting one ormore, such as all, of the 29 isoallotypes of human IgG provided in FIG.29 using polyclonal antibodies, e.g., anti-IgG. In various embodiments,the methods include detecting shortcomings in one or more assays, suchas assays applying monoclonal anti-IgG as described herein. As such, themethods also include detecting the absence of one or more of the 29isoallotypes of human IgG provided in FIG. 29 from a panel usingmonoclonal antibodies, e.g., anti-IgG. In such aspects, monoclonalanti-IgG does not bind to all of the 29 isoallotypes, e.g., only bindsto less than the 29 isoallotypes, such as 24 out of 29 isoallotypes. Invariations of the methods, the monoclonal anti-IgG assay can fail torecognize IgG3-03, IgG3-13, IgG4-01, IgG4-02, and IgG4-03 (see FIG. 27)and the methods include detecting each failed recognition.

In FIG. 29, amino acid variations among IgG subclasses and theirrespective isoallotypes are shown along the top, broken up by the regionin which the mutation occurs (CH1, hinge, CH2 or CH3; labeled in darkgrey) as well as by those variations that occur only between subclassesthemselves (amino acid residue not highlighted) or variations thatdefine the isoallotypes (amino acid position highlighted with darkenedshade of grey). Specific changes within an IgG subclass are shown asunderlined. While all of the IgG1, IgG2, and IgG4 isoallotypes havetheir own hinge regions, these do not change in their respectivevariants. However, the IgG3 hinge is variable among isoallotypes; withthe presence or absence of specific hinge sequences shown in the tableprovided in FIG. 29. The ability of each isoallotype to be recognized byeach of the tested anti-IgG reagents is indicated with a + or on theright side of the table. (EU numbering scheme used for amino acidposition.) For each IgG subtype, the *01 designation is the canonicalsequence. Furthermore, as provided in FIG. 29, IGHG1*01v2 is equivalentto IGHG1*05 and IGHG1*04v2 is equivalent to IGHG1*06.

Polypeptides

The term “polypeptide” or “peptide” refers to a polymer of amino acidswithout regard to the length of the polymer; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not specify or exclude post-expressionmodifications of polypeptides, for example, polypeptides which includethe covalent attachment of glycosyl groups, acetyl groups, phosphategroups, lipid groups and the like are expressly encompassed by the termpolypeptide. Also included within the definition are polypeptides whichcontain one or more analogs of an amino acid (including, for example,non-naturally occurring amino acids, amino acids which only occurnaturally in an unrelated biological system, modified amino acids frommammalian systems etc.), polypeptides with substituted linkages, as wellas other modifications known in the art, both naturally occurring andnon-naturally occurring.

The term “isolated protein,” “isolated polypeptide,” or “isolatedpeptide” is a protein, polypeptide or peptide that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, a peptidethat is chemically synthesized or synthesized in a cellular systemdifferent from the cell from which it naturally originates will be“isolated” from its naturally associated components. A protein can alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.

The terms “polypeptide”, “protein”, “peptide,” “antigen,” or “antibody”within the meaning of the present invention, includes variants, analogs,orthologs, homologs and derivatives, and fragments thereof that exhibita biological activity, generally in the context of being able to inducean immune response in a subject, or bind an antigen in the case of anantibody.

The polypeptides of the invention include an amino acid sequence derivedfrom Kell system antigens or fragments thereof, corresponding to theamino acid sequence of a naturally occurring protein or corresponding tovariant protein, i.e., the amino acid sequence of the naturallyoccurring protein in which a small number of amino acids have beensubstituted, added, or deleted but which retains essentially the sameimmunological properties. In addition, such derived portion can befurther modified by amino acids, especially at the N- and C-terminalends to allow the polypeptide or fragment to be conformationallyconstrained and/or to allow coupling to an immunogenic carrier afterappropriate chemistry has been carried out. The polypeptides of thepresent invention encompass functionally active variant polypeptidesderived from the amino acid sequence of Kell system antigens in whichamino acids have been deleted, inserted, or substituted withoutessentially detracting from the immunological properties thereof, i.e.such functionally active variant polypeptides retain a substantialpeptide biological activity.

In one embodiment, such functionally active variant polypeptides exhibitat least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity toan amino acid sequence of the blood group antigens disclosed herein.Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). An alternative algorithm when comparing a sequenceof the invention to a database containing a large number of sequencesfrom different organisms is the computer program BLAST, especiallyblastp or tblastn, using default parameters. See, e.g., Altschul et al.,J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997).

Functionally active variants include naturally occurring functionallyactive variants such as allelic variants and species variants andnon-naturally occurring functionally active variants that can beproduced by, for example, mutagenesis techniques or by direct synthesis.

A functionally active variant can exhibit, for example, at least 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acidsequence of a Kell system or other antigen disclosed herein, and yetretain a biological activity. Where this comparison requires alignment,the sequences are aligned for maximum homology. The site of variationcan occur anywhere in the sequence, as long as the biological activityis substantially similar to the Kell system or other antigens disclosedherein, e.g., ability to induce a tolerance response. Guidanceconcerning how to make phenotypically silent amino acid substitutions isprovided in Bowie et al., Science, 247: 1306-1310 (1990), which teachesthat there are two main strategies for studying the tolerance of anamino acid sequence to change. The first strategy exploits the toleranceof amino acid substitutions by natural selection during the process ofevolution. By comparing amino acid sequences in different species, theamino acid positions which have been conserved between species can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions in which substitutionshave been tolerated by natural selection indicate positions which arenot critical for protein function. Thus, positions tolerating amino acidsubstitution can be modified while still maintaining specificimmunogenic activity of the modified polypeptide.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site-directed mutagenesis oralanine-scanning mutagenesis can be used (Cunningham et al., Science,244: 1081-1085 (1989)). The resulting variant polypeptides can then betested for specific biological activity.

According to Bowie et al., these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,the most buried or interior (within the tertiary structure of theprotein) amino acid residues require nonpolar side chains, whereas fewfeatures of surface or exterior side chains are generally conserved.

Methods of introducing a mutation into amino acids of a protein is wellknown to those skilled in the art. (See, e. g., Ausubel (ed.), CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989)).

Mutations can also be introduced using commercially available kits suchas “QuikChange Site-Directed Mutagenesis Kit” (Stratagene) or directlyby peptide synthesis. The generation of a functionally active variant toan peptide by replacing an amino acid which does not significantlyinfluence the function of said peptide can be accomplished by oneskilled in the art.

A type of amino acid substitution that can be made in the polypeptidesof the invention is a conservative amino acid substitution. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity can be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See e.g. Pearson, Methods Mol.Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Variousconservative amino acids substitution groups include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992). A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

A functionally active variant can also be isolated using a hybridizationtechnique. Briefly, DNA having a high homology to the whole or part of anucleic acid sequence encoding the peptide, polypeptide or protein ofinterest, e.g. Kell system antigens, is used to prepare a functionallyactive peptide. Therefore, a polypeptide of the invention also includesentities which are functionally equivalent and which are encoded by anucleic acid molecule which hybridizes with a nucleic acid encoding anyone of the Kell system antigens or a complement thereof. One of skill inthe art can easily determine nucleic acid sequences that encode peptidesof the invention using readily available codon tables. As such, thesenucleic acid sequences are not presented herein.

Nucleic acid molecules encoding a functionally active variant can alsobe isolated by a gene amplification method such as PCR using a portionof a nucleic acid molecule DNA encoding a peptide, polypeptide, protein,antigen, or antibody of interest, e.g. Kell system antigens, as theprobe.

For the purpose of the present invention, it should be considered thatseveral polypeptides or antigens of the invention can be used incombination. All types of possible combinations can be envisioned. Thesame sequence can be used in several copies on the same polypeptidemolecule, or wherein peptides of different amino acid sequences are usedon the same polypeptide molecule; the different peptides or copies canbe directly fused to each other or spaced by appropriate linkers. Asused herein the term “multimerized (poly)peptide” refers to both typesof combination wherein polypeptides of either different or the sameamino acid sequence are present on a single polypeptide molecule. From 2to about 20 identical and/or different peptides can be thus present on asingle multimerized polypeptide molecule.

In one embodiment of the invention, a peptide, polypeptide, protein, orantigen of the invention is derived from a natural source and isolatedfrom a bacterial source. A peptide, polypeptide, protein, or antigen ofthe invention can thus be isolated from sources using standard proteinpurification techniques.

Alternatively, peptides, polypeptides and proteins of the invention canbe synthesized chemically or produced using recombinant DNA techniques.For example, a peptide, polypeptide, or protein of the invention can besynthesized by solid phase procedures well known in the art. Suitablesyntheses can be performed by utilising “T-boc” or “F-moc” procedures.Cyclic peptides can be synthesized by the solid phase procedureemploying the well-known “F-moc” procedure and polyamide resin in thefully automated apparatus. Alternatively, those skilled in the art willknow the necessary laboratory procedures to perform the processmanually. Techniques and procedures for solid phase synthesis aredescribed in ‘ Solid Phase Peptide Synthesis: A Practical Approach’ byE. Atherton and R. C. Sheppard, published by IRL at Oxford UniversityPress (1989) and ‘Methods in Molecular Biology, Vol. 35: PeptideSynthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7, pp91-171 by D. Andreau et al.

Alternatively, a polynucleotide encoding a peptide, polypeptide orprotein of the invention can be introduced into an expression vectorthat can be expressed in a suitable expression system using techniqueswell known in the art, followed by isolation or purification of theexpressed peptide, polypeptide, or protein of interest. A variety ofbacterial, yeast, plant, mammalian, and insect expression systems areavailable in the art and any such expression system can be used.Optionally, a polynucleotide encoding a peptide, polypeptide or proteinof the invention can be translated in a cell-free translation system.

Nucleic acid sequences corresponding to Kell system antigens can also beused to design oligonucleotide probes and used to screen genomic or cDNAlibraries for genes encoding other variants or from other species. Thebasic strategies for preparing oligonucleotide probes and DNA libraries,as well as their screening by nucleic acid hybridization, are well knownto those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I,supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,supra; Sambrook et al., supra. Once a clone from the screened libraryhas been identified by positive hybridization, it can be confirmed byrestriction enzyme analysis and DNA sequencing that the particularlibrary insert contains a Kell system antigen gene, or a homologthereof. The genes can then be further isolated using standardtechniques and, if desired, PCR approaches or restriction enzymesemployed to delete portions of the full-length sequence.

Alternatively, DNA sequences encoding the proteins of interest can beprepared synthetically rather than cloned. The DNA sequences can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select codons for the intended host ifthe sequence will be used for expression. The complete sequence isassembled from overlapping oligonucleotides prepared by standard methodsand assembled into a complete coding sequence. See, e.g., Edge (1981)Nature 292: 756; Nambair et al. (1984) Science 223: 1299; Jay et al.(1984) J. Biol. Chem. 259: 6311.

Once coding sequences for the desired proteins have been prepared orisolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pUC1 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra. The gene can be placed under the control of a promoter,ribosome binding site (for bacterial expression) and, optionally, anoperator (collectively referred to herein as “control” elements), sothat the DNA sequence encoding the desired protein is transcribed intoRNA in the host cell transformed by a vector containing this expressionconstruction. The coding sequence can or cannot contain a signal peptideor leader sequence. Leader sequences can be removed by the host inpost-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397. Examples of vectors include pET32a(+) andpcDNA3002Neo.

Other regulatory sequences can also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements can also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences can be ligated tothe coding sequence prior to insertion into a vector, such as thecloning vectors described above. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

In some cases it can be necessary to modify the coding sequence so thatit can be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It can also bedesirable to produce mutants or analogs of the protein. Mutants oranalogs can be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are describedin, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, HEK293F cells, NSO-1 cells, aswell as others. Similarly, bacterial hosts such as E. coli, Bacillussubtilis, and Streptococcus spp., will find use with the presentexpression constructs. Yeast hosts useful in the present inventioninclude, but are not limited to, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, but are not limited to,Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by culturing host cells transformedby an expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. The selection of the appropriate growthconditions and recovery methods are within the skill of the art.

Kell system antigen protein sequences can also be produced by chemicalsynthesis such as solid phase peptide synthesis, using known amino acidsequences or amino acid sequences derived from the DNA sequence of thegenes of interest. Such methods are known to those skilled in the art.See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis,2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E.Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp.3-254, for solid phase peptide synthesis techniques; and M. Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis. Chemicalsynthesis of peptides can be performed if a small fragment of theantigen in question is capable of raising an immunological response inthe subject of interest.

Polypeptides of the invention can also include those that arise as aresult of the existence of multiple genes, alternative transcriptionevents, alternative RNA splicing events, and alternative translationaland postranslational events. A polypeptide can be expressed in systems,e.g. cultured cells, which result in substantially the samepostranslational modifications present as when the peptide is expressedin a native cell, or in systems that result in the alteration oromission of postranslational modifications, e.g. glycosylation orcleavage, present when expressed in a native cell.

A peptide, polypeptide, protein, or antigen of the invention can beproduced as a fusion protein that contains other distinct amino acidsequences that are not part of the Kell system antigen sequencesdisclosed herein, such as amino acid linkers or signal sequences orimmunogenic carriers, as well as ligands useful in protein purification,such as glutathione-S-transferase, histidine tag, and staphylococcalprotein A. More than one polypeptide of the invention can be present ina fusion protein. The heterologous polypeptide can be fused, forexample, to the N-terminus or C-terminus of the peptide, polypeptide orprotein of the invention. A peptide, polypeptide, protein, or antigen ofthe invention can also be produced as fusion proteins includinghomologous amino acid sequences.

Blood Group Antigen Proteins

Any of a variety of cell surface proteins found on red blood cells canbe used in the practice of the present invention. In one embodiment, theproteins are blood group antigens, such as the Kell system antigens.Information on such antigens and, in particular, soluble forms areavailable in the art, for example, in Ridgwell et al., TransfusionMedicine, 17: 384-394 (2007).

Kell (CD238) is a clinically important human blood group antigen systemincluding 28 antigens (Daniels et al., 2007, International Society ofBlood Transfusion Committee on Terminology for Red Cell SurfaceAntigens: Cape Town report. Vox Sanguinis, 92, 250-253). The Kellantigens are carried by a single pass type II (cytoplasmic N-terminus)red blood cell membrane glycoprotein. The Kell glycoprotein is expressedin red cells and haematopoietic tissue (bone marrow and foetal liver)and to a lesser extent in other tissues, including brain, lymphoidorgans, heart and skeletal muscle (Russo et al., 2000, Blood, 96,340-346). The K/k (KEL1/KEL2) blood group antigen polymorphism isdetermined by a single nucleotide polymorphism (SNP) resulting in thepresence of methionine (M) or threonine (T), respectively, at amino acid193 of the extracellular C-terminal domain (Lee, 1997, Vox Sanguinis,73, 1-11). The other mostclinically significant antithetical antigensKp^(a)/Kp^(b) (KEL3/KEL4) and Js^(a)/Js^(b) (KEL6/KEL7) are also theresult of SNPs resulting in single amino acid changes in theextracellular domain (Lee, 1997, Vox Sanguinis, 73, 1-11).

Kell system antibodies are known to cause haemolytic transfusionreactions and haemolytic disease of the fetus and newborn (HDFN).Kell-related HDFN may be because of suppression of fetal erythropoiesisin addition to immune destruction of red blood cells as in most othercases of HDFN (Vaughan et al., 1998, New England Journal of Medicine,338, 798-803; Daniels et al., 2003, Transfusion, 43, 115-116). Anti-K(KEL1) is the most commonly encountered immune red cell antibody outsidethe ABO and Rh systems, and other antigens of the Kell blood groupsystem, e.g. k (KEL2), Kp^(a) (KEL3), Kp^(b) (KEL4), Js^(a) (KEL6) andJs^(b) (KEL7) are also capable of stimulating the production ofhaemolytic antibodies and causing HDFN (Daniels, 2002, Human BloodGroups (2nd edn). Blackwell, Oxford).

The Duffy (Fy, CD234) blood group antigens are carried by a type IIImembrane glycoprotein, which is predicted to span the membrane seventimes with a glycosylated extracellular N-terminus and a cytoplasmicC-terminus. It is expressed in red blood cells, vascular endothelialcells and a wide range of other tissues including kidney, lung, liver,spleen, brain (Iwamoto et al., 1996, Blood, 87, 378-385) and colon(Chaudhuri et al., 1997, Blood, 89, 701-712). The Fy^(a)/Fy^(b)(FY1/FY2) blood group polymorphorism is determined by an SNP resultingin the presence of glycine (G) or aspartic acid (D), respectively, atamino acid 42 in the N-terminal extracellular domain (Iwamoto et al.,1995, Blood, 85, 622-626; Mallinson et al., 1995, British Journal ofHaematology, 90, 823-82; Tournamille et al., 1995, Human Genetics, 95,407-410). Duffy blood group system antibodies can cause haemolytictransfusion reactions (Boyland et al., 1982, Transfusion, 22, 402;Sosler et al., 1989, Transfusion, 29, 505-507) and HDFN (Vescio et al.,1987, Transfusion, 27, 366; Goodrick et al., 1997, Transfusion Medicine,7, 301-304).

The Lutheran (Lu, B-CAM, CD239) blood group antigens are carried by twosingle-pass type I (cytoplasmic C-terminus) membrane glycoproteins,which differ in the length of their cytoplasmic domains [the B-CAMglycoprotein has a shorter C-terminal cytoplasmic tail than Lu (Campbellet al., 1994, Cancer Research, 54, 5761-5765)]. The Lu glycoprotein hasfive extracellular immunoglobulin-like domains and is a member of theimmunoglobulin gene superfamily (IgSF) (Parsons et al., 1995,Proceedings of the National Academy of Science of the United States ofAmerica, 92, 5496-5500) and is expressed in red blood cells and a widerange of other tissues (Reid & Lomas-Francis, 2004, The Blood GroupAntigens Factsbook (2nd edn). Academic Press, London). The Lu^(a)/Lu^(b)(LU1/LU2) blood group antigen polymorphism is determined by a SNPresulting in the presence of histidine (H) or arginine (R),respectively, at amino acid 77 of the first predicted N-terminal IgSFdomain (El Nemer et al., 1997). Lutheran blood group system antibodieshave been reported to be involved in mild delayed haemolytic transfusionreactions (Daniels, 2002, Human Blood Groups (2nd edn). Blackwell,Oxford) but are rarely involved in HDFN (Inderbitzen et al., 1982,Transfusion, 22, 542).

Antibodies

As used herein, the term “antibody” refers to any immunoglobulin orintact molecule as well as to fragments thereof that bind to a specificepitope. Such antibodies include, but are not limited to polyclonal,monoclonal, chimeric, humanized, single chain, Fab, Fab′, F(ab)′fragments and/or F(v) portions of the whole antibody and variantsthereof. All isotypes are encompassed by this term, including IgA, IgD,IgE, IgG, and IgM.

As used herein, the term “antibody fragment” refers specifically to anincomplete or isolated portion of the full sequence of the antibodywhich retains the antigen binding function of the parent antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

An intact “antibody” includes at least two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chain iscomposed of a heavy chain variable region (abbreviated herein as HCVR orV_(H)) and a heavy chain constant region. The heavy chain constantregion is composed of three domains, CH₁, CH₂ and CH₃. Each light chainis composed of a light chain variable region (abbreviated herein as LCVRor V_(L)) and a light chain constant region. The light chain constantregion is composed of one domain, C_(L). The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyl-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. The term antibody includesantigen-binding portions of an intact antibody that retain capacity tobind. Examples of binding include (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment including two Fab fragments linkedby a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAbfragment (Ward et al., Nature, 341:544-546 (1989)), which consists of aVH domain; and (vi) an isolated complementarity determining region(CDR).

As used herein, the term “single chain antibodies” or “single chain Fv(scFv)” refers to an antibody fusion molecule of the two domains of theFv fragment, V_(L) and V_(H). Although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see, e.g., Bird et al., Science, 242:423-426 (1988); and Hustonet al., Proc Natl Acad Sci USA, 85:5879-5883 (1988)). Such single chainantibodies are included by reference to the term “antibody” fragmentsand can be prepared by recombinant techniques or enzymatic or chemicalcleavage of intact antibodies.

As used herein, the term “human sequence antibody” includes antibodieshaving variable and constant regions (if present) derived from humangermline immunoglobulin sequences. The human sequence antibodies of theinvention can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo). Suchantibodies can be generated in non-human transgenic animals, e.g., asdescribed in PCT App. Pub. Nos. WO 01/14424 and WO 00/37504. However,the term “human sequence antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (e.g., humanized antibodies).

Also, recombinant immunoglobulins can be produced. See, Cabilly, U.S.Pat. No. 4,816,567, incorporated herein by reference in its entirety andfor all purposes; and Queen et al., Proc Natl Acad Sci USA,86:10029-10033 (1989).

As used herein, the term “monoclonal antibody” refers to a preparationof antibody molecules of single molecular composition. A monoclonalantibody composition displays a single binding specificity and affinityfor a particular epitope. Accordingly, the term “human monoclonalantibody” refers to antibodies displaying a single binding specificitywhich have variable and constant regions (if present) derived from humangermline immunoglobulin sequences. In one aspect, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome including a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

As used herein, the term “antigen” refers to a substance that promptsthe generation of antibodies and can cause an immune response. It can beused interchangeably in the present disclosure with the term“immunogen”. In the strict sense, immunogens are those substances thatelicit a response from the immune system, whereas antigens are definedas substances that bind to specific antibodies. An antigen or fragmentthereof can be a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein can inducethe production of antibodies (i.e., elicit the immune response), whichbind specifically to the antigen (given regions or three-dimensionalstructures on the protein).

An “epitope” refers to the portion of the antigen bound by an antibody.Antigens can include multiple epitopes. Where the antigen is a protein,linear epitopes can range from about 5 to 20 amino acids in length.Antibodies can also recognize conformational determinants formed bynon-contiguous residues on an antigen, and an epitope can thereforerequire a larger fragment of the antigen to be present for binding, e.g.a protein domain, or substantially all of a protein sequence. It willtherefore be appreciated that a protein, which can be several hundredamino acids in length, can include a number of distinct epitopes.

As used herein, the term “humanized antibody,” refers to at least oneantibody molecule in which the amino acid sequence in the non-antigenbinding regions and/or the antigen-binding regions has been altered sothat the antibody more closely resembles a human antibody, and stillretains its original binding ability.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., Proc Natl Acad Sci, 81:6851-6855 (1984),incorporated herein by reference in their entirety) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. For example, the genes from a mouseantibody molecule specific for an autoinducer can be spliced togetherwith genes from a human antibody molecule of appropriate biologicalactivity. A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine mAb and a human immunoglobulinconstant region.

In addition, techniques have been developed for the production ofhumanized antibodies (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,225,539,which are incorporated herein by reference in their entirety). Animmunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, referredto as complementarity determining regions (CDRs). Briefly, humanizedantibodies are antibody molecules from non-human species having one ormore CDRs from the non-human species and a framework region from a humanimmunoglobulin molecule.

Alternatively, techniques described for the production of single chainantibodies can be adapted to produce single chain antibodies against animmunogenic conjugate of the present disclosure. Single chain antibodiesare formed by linking the heavy and light chain fragments of the Fvregion via an amino acid bridge, resulting in a single chainpolypeptide. Fab and F(ab′)2 portions of antibody molecules can beprepared by the proteolytic reaction of papain and pepsin, respectively,on substantially intact antibody molecules by methods that arewell-known. See e.g., U.S. Pat. No. 4,342,566. Fab′ antibody moleculeportions are also well-known and are produced from F(ab′)2 portionsfollowed by reduction of the disulfide bonds linking the two heavy chainportions as with mercaptoethanol, and followed by alkylation of theresulting protein mercaptan with a reagent such as iodoacetamide.

Antibody Assays

A number of screening assays are known in the art for assayingantibodies of interest to confirm their specificity and affinity and todetermine whether those antibodies cross-react with other proteins.

The terms “specific binding” or “specifically binding” refer to theinteraction between the antigen and their corresponding antibodies. Theinteraction is dependent upon the presence of a particular structure ofthe protein recognized by the binding molecule (i.e., the antigen orepitope). In order for binding to be specific, it should involveantibody binding of the epitope(s) of interest and not backgroundantigens.

Once antibodies are produced, they are assayed to confirm that they arespecific for the antigen of interest and to determine whether theyexhibit any cross reactivity with other antigens. One method ofconducting such assays is a sera screen assay as described in U.S. App.Pub. No. 2004/0126829, the contents of which are hereby expresslyincorporated herein by reference. However, other methods of assaying forquality control are within the skill of a person of ordinary skill inthe art and therefore are also within the scope of the presentdisclosure.

Antibodies, or antigen-binding fragments, variants or derivativesthereof of the present disclosure can also be described or specified interms of their binding affinity to an antigen. The affinity of anantibody for an antigen can be determined experimentally using anysuitable method. (See, e.g., Berzofsky et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992); and methods described herein). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions (e.g., salt concentration, pH).Thus, measurements of affinity and other antigen-binding parameters(e.g., K_(D), K_(a), K_(d)) can be made with standardized solutions ofantibody and antigen, and a standardized buffer.

The affinity binding constant (K_(aff)) can be determined using thefollowing formula:

$K_{aff} = \frac{\left( {n - 1} \right)}{2\left( {{n\left\lbrack {mAb}^{\prime} \right\rbrack}_{t} = \lbrack{mAb}\rbrack_{t}} \right)}$

in which:

$n = \frac{\lbrack{mAg}\rbrack_{t}}{\left\lbrack {mAg}^{\prime} \right\rbrack_{t}}$

[mAb] is the concentration of free antigen sites, and [mAg] is theconcentration of free monoclonal binding sites as determined at twodifferent antigen concentrations (i.e., [mAg]_(t) and [mAg]_(t)) (Beattyet al., J Imm Meth, 100:173-179 (1987)).

The term “high affinity” for an antibody refers to an equilibriumassociation constant (K_(aff)) of at least about 1×10⁷ liters/mole, orat least about 1×10⁸ liters/mole, or at least about 1×10⁹ liters/mole,or at least about 1×10¹⁰ liters/mole, or at least about 1×10¹¹liters/mole, or at least about 1×10¹² liters/mole, or at least about1×10¹³ liters/mole, or at least about 1×10¹⁴ liters/mole or greater.“High affinity” binding can vary for antibody isotypes. K_(D), theequilibrium dissociation constant, is a term that is also used todescribe antibody affinity and is the inverse of K_(aff).

K_(D), the equilibrium dissociation constant, is a term that is alsoused to describe antibody affinity and is the inverse of K_(aff). IfK_(D) is used, the term “high affinity” for an antibody refers to anequilibrium dissociation constant (K_(D)) of less than about 1×10⁻⁷mole/liters, or less than about 1×10⁻⁸ mole/liters, or less than about1×10⁻⁹ mole/liters, or less than about 1×10⁻¹⁰ mole/liters, or less thanabout 1×10⁻¹¹ mole/liters, or less than about 1×10⁻¹² mole/liters, orless than about 1×10⁻¹³ mole/liters, or less than about 1×10⁻¹⁴mole/liters or lower.

The immunoglobulin molecules of the present invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2), or subclass of immunoglobulin molecule. In someembodiments, the antibodies are antigen-binding antibody fragments(e.g., human) and include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments including either a V_(L) orV_(H) domain. Antigen-binding antibody fragments, including single-chainantibodies, can include the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, CH1, CH2,and CH3 domains. Also included in the present disclosure areantigen-binding fragments including any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains.

Kits

The invention provides kits including antibodies produced in accordancewith the present disclosure which can be used, for instance, for theapplications described above. The article of manufacture includes acontainer with a label. Suitable containers include, for example,bottles, vials, and test tubes. The containers can be formed from avariety of materials such as glass or plastic. The container holds acomposition which includes an active agent that is effective forapplications, such as those described above. The active agent in thecomposition can include antibodies. The label on the container indicatesthat the composition is used for a particular application, and can alsoindicate directions for use, such as those described above.

Utility

Various methodologies exist for screening patients for anti-RBCalloantibodies; however, each method has a common theme. Samples fromthe patient (serum or plasma) is incubated with a panel of RBCs thatexpress the common RBC alloantigens (screening cells) and if the patienthas alloantibodies, then they bind to the screening cells (see FIG. 13).However, most non-ABO alloantibodies are IgG, and are not directlyagglutinating. Thus, to facilitate detection, an additional antibody(anti-human globulin [AHG]) is added, to crosslink the patients IgG (seeFIG. 13). Non-agglutination based assays (e.g. gel card, solid phase,flow cytometry, etc.) likewise use AHG based binding and/or detectionsystems.

Human IgGs come in 4 distinct forms, each of which has a separate heavychain (IgG1, IgG2, IgG3, and IgG4). In most patients, anti-RBCalloantibodies are a mixture of each of these forms; however, in somepatients only some IgG subtypes are present, and in rare patients, onlya single type of IgG is detectable. Thus, the ability of AHG torecognize each of the IgG subtypes is required in order to achieve theability to uniformly detect anti-RBC alloantibodies. However, no pureanti-RBC alloantibodies of different IgG subtypes are available toassess the ability of AHG to bind each of the subtypes. Since most AHGis polyclonal antisera (often from rabbits), it will vary from batch tobatch, both due to differences in response of individual animals andalso due to the changing nature of immune responses in a given animal.Moreover, use of the same AHG will vary in different diagnosticplatforms and using different methods. Thus, at the current time, thepresence of an antibody that is not recognized by AHG is a source offalse negative patient screens prior to transfusion.

Furthermore, the subject embodiments demonstrate “blind spots” inisoalloantibody detection by a monoclonal anti-IgG. Should a patienthave anti-RBC antibodies predominantly of an IgG3 subtype of the IgG3-03and/or IgG3-13 variety, it is possible that a clinically significantalloantibody would be missed. IgG-03 and IgG-13 are estimated at afrequency of 1-3% of Caucasian and 20-30% of certain Africanpopulations. The non-reactivity of, for example, IgG4 isoallotypes hasnot been previously reported.

The subject matter of the present disclosure addresses these and otherproblems.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1: Generation of Monoclonal Antibodies Against Kell Antigens

Mice expressing the human Kell glycoprotein (K variant) on RBCs weregenerated. Transgenic RBCs were then transfused into wild-type mice,thus allowing cell surface expression without the introduction ofadditional antigens. The recipient mice were pretreated with poly (I:C),which acts a an adjuvant to increase antibody responses to antigens ontransfused RBCs, as first described by Dr. Zimring (Transfusion 46(9):1526-36, 2006). Splenocytes from immunized mice were fused with myelomapartners and monoclonal antibodies were isolated. Three clones wereisolated that produce monoclonal IgG antibodies, which recognize the Kform of the Kell glycoprotein but not the k form. These antibodies areuseful typing reagents for human RBCs by a variety of methods,including, but not limited to, fluid phase agglutination, solid phasedetection, tube gel detection, flow cytometry detection, enzyme linkedimmunoadsorbant assay, radioimmunoassay, and Western blot.

Shown in FIGS. 1-6 are the sequences of the antibodies obtained. Uponsequencing, it was determined that the antibodies designated PUMA 1 andPUMA 2 were the same. The shading indicates where the highly variableregions begin. The CDR regions of each heavy or light chain areunderlined.

The specificities of the antibodies are shown in FIGS. 7-10. Thespecificities of the antibodies were determined to be: PUMA1/2 (KEL1 orK), PUMA 3 (a common Kell epitope, PUMA 4 (KEL4 or Kp^(b)). Flowcytometry was utilized to test antibody specificity by indirectimmunofluorescence, using the monocolonal antibodies as the primaryreagent and a goat-anti-mouse antibody (conjugated to allophycocyanin)as a secondary antibody. Different target cells expressing differentKell variants were used to determine specificity. Targets included RBCsthat phenotyped as homozygous for the 3 main antithetical antigens inthe Kell system, K/K, k/k, Kp^(b)/Kp^(b), Kp^(a)/Kp^(a). Js^(b)/Js^(b),Js^(a)/Js^(a). Differential binding to such tarets tests specificity. Inthe case of PUMA 1/2, binding was only observed when K was present butnot on k/k RBCs. In the case of PUMA 3, binding was observed on all RBCsregardless of phenotype for K/k, Kp^(a)/Kp^(b), or Js^(a)/Js^(b), thusindicating a common epitope outside these systems. However, PUMA 3 boundto only KELL glycoprotein transgenic murine RBCs and not wild-typemurine RBCs; thus, the epitope recognized by PUMA 3 is on the KELLmolecule, but not K/k, Kp^(a)/Kp^(b), or Js^(a)/Js^(b). For PUMA 4,binding was only observed when Kp^(b) was present but not onKp^(a)/Kp^(a) RBCs.

Example 2: Antibody Modification

To allow engineering and manipulation of PUMA1, rapid amplification ofcDNA ends (RACE) was performed on both the heavy and light chains ofPUMA1, and the sequence for the PUMA1 antibody was elucidated (see FIGS.1 and 2). Based upon the predicted sequence, mass spectrometry wasperformed on purified monoclonal PUMA1 and predicted peptides wereconfirmed for both the heavy and light chain, demonstrating that thecorrect cDNA was amplified. The identified sequence of PUMA1 heavy chainwas cloned in frame with cDNA coding sequence for the mouse IgG3subtypes, in a eukaryotic expression vector. Similarly, the sequence ofthe PUMA1 light chain was cloned into a Eukaryotic expression vector.IgG3 was chosen, since it is typically known to have a diminishedcapacity to induce clearance of bound targets than IgG2a. The plasmidencoding PUMA1 IgG3 heavy chain was transfected into CHO cells, alongwith the expression vector for light chain, and PUMA1 IgG3 was thenpurified from culture supernatant using protein A affinitychromatography. Recombinant PUMA1 IgG2a was engineered and expressed inthe same way, to allow PUMA1 IgG2a expressed in the same system as thePUMA1 IgG3. Similar to the above murine sequences, PUMA1 has now beenhumanized by recombinant fusion of the CDRs with human IgG1, IgG2, IgG3and IgG4 (FIG. 11). An example of the expression of humanizedantibodies, while maintaining ability to bind RBCs is shown in FIG. 12.

Example 3: Evaluation of PUMA1 IgG1, IgG2, IgG3, and IgG4 Binding toAnti-Human Globulin (AHG)

According to the subject aspects, pure PUMA1 IgG1, IgG2, IgG3, and IgG4were generated and were used to evaluate various AHG preparations, asdemonstrated by use in various platforms. FIG. 15 shows the use of PUMA1IgG1, IgG2, IgG3, and IgG4 to evaluate various AHG preparations using aFACS assay. FIG. 16 shows an evaluation using a gel testing assay. FIGS.17-22 show an evaluation using a using a tube testing assay. FIGS. 23-25show an evaluation using a solid phase testing assay. Significantly, asshown in FIG. 15, the Immucor monoclonal AHG did not detect IgG4—as isdescribed in limitations of the Immucor AHG; thus, this validates thedisclosure's utility to detect known AHG defects.

Example 4: Evaluation of Serological Blind-Spots for Variants of HumanImmunoglobulins by Anti-Immunoglobulin Reagents

Alloantibodies to non-ABO red blood cell (RBC) antigens are usually notdirect agglutinins, and typically require the addition of anti-IgG tofacilitate their detection. The two current methods of manufacturingreagent grade anti-IgG consist of either generating polyclonal anti-IgGfrom serum of animals (typically rabbits) immunized with polyclonalhuman IgG or the use of monoclonal antibody based reagents, typicallyderived from murine sources. While polyclonal anti-IgG contains multiplespecificities, and can be a very sensitive reagent, it suffers thepotential for variation from animal to animal and from batch to batch.In contrast, monoclonal antibodies are a stable and consistent reagent;however, they can display a more narrow range of reactivity, astypically a single epitope is recognized on the target immunoglobulin.

There are four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4). There isalso genetic variation within IgG subtypes wherein the genetic variantsare called isoallotypes. There are at least 29 different isoallotypes,with 7, 4, 15, and 3 isoallotypes provided for IgG1-4, respectively.While anti-IgG reagents are required to meet specifications andstandards for licensure, the reactivity of anti-IgG with differentisoallotypes has not before been characterized. As such, the reactivityof anti-IgG with different isoallotypes is evaluated according to thesubject embodiments.

In various aspects, the methods include identifying positive and/ornegative reactivity of anti-IgG with one or more isoallotypes, such asany one or combination of the isoallotypes provided in FIG. 29 and/orcharacterizing the one or more isoallotypes according to theidentification. In various aspects, the methods also include identifyingthe amount of positive and/or negative reactivity of anti-IgG with oneor more isoallotypes and/or characterizing the one or more isoallotypesaccording to the identification.

The generation of test reagents to assess specificity of anti-IgG hashistorically been challenging and difficult to standardize. In someaspects, solid media is coated with purified IgG of different types todetermine anti-IgG reactivity (e.g. ELISAs). While meaningful, suchapproaches are outside of the context in which IAT and DAT tests are run(e.g. testing IgG bound to the surface of RBCs), and thus may notreflect anti-IgG performance for clinical testing. To create the abilityto characterize anti-IgG sensitivities and specificities in the contextin which anti-IgG is used in immunohematology, a new monoclonal antibodywas generated against the K antigen and isolated the cDNA sequence forthe heavy and light chain variable regions. The heavy chain variableregion was then ligated into expression vectors using a strategy thatfused it in frame with the constant region of human IgG. Separateexpression vectors for each of the 29 known IgG isoallotypes werecreated, allowing expression and purification of isoallotypic variants.This approach isolates isoallotypic variation as an independentvariable, as the antigen binding domain is the same for each anti-Kvariant.

The panel of 29 isoallotypes was applied according to the subjectaspects to characterize the performance of different, anti-IgG reagents.Herein it is provided that one or more monoclonal anti-IgG fails torecognize two particular isoallotypes of IgG3 (IgG3-03 and IgG3-13). Inaddition, it was confirmed that the known property of such a monoclonalanti-IgG does not react with canonical IgG4, and also observed that thisnon-reactivity extends to the 3 known isoallotypes of IgG4. IgG4 is notknown to typically result in acute hemolytic transfusion reactions, andthus non-reactivity with IgG4 is not typically considered as a weaknessof monoclonal anti-IgG. However, IgG3 is often considered the mosthemolytic IgG subclass. The identified IgG3 isoallotypes have asignificant frequency in certain populations, including humans native toAfrica.

1. Materials and Methods

A. Mice:

K transgenic mice (published as KEL1 mice) were generated andcharacterized as previously described and were bred in the BloodworksNWvivarium⁴. CBy.RBF-Rb(8.12)5Bnr/J mice were purchased from Jackson Labs,Bar Harbor Me. (Cat #001802). All mice were maintained on standardrodent chow and water in a temperature- and light-controlledenvironment. All experiments were performed according to approvedInstitutional Animal Care and Use Committee (IACUC) procedures.

B. Immunizations and Isolation of Monoclonal Antibody:

RBCs were obtained by peripheral blood from K mice and were transfusedinto CByJ.RBF-Rb(8.12)5Bnr/J recipients by lateral tail vein injection.CByJ.RBF-Rb(8.12)5Bnr/J recipients were treated with poly (I:C) prior totransfusion as an adjuvant, as previously described.⁵ Alloimmunizationto the K antigen was monitored by analyzing sera from transfused mice,using K+ RBCs as targets, and performing indirect immunofluorescence byflow cytometry (see below). Immunized mice were boosted 3 days prior tosacrifice, and then splenocytes were harvested and fused (usingpolyethylene glycol) to myeloma partner FOX-NY (ATCC CRL-1732) followedby culturing in selective media by routine methods. Clones were isolatedby limiting dilution culture techniques, and supernatants were screenedfor anti-K using indirect immunofluorescence and flow cytometry.

C. Identification and Synthesis of Heavy Chain and Light Chain VariableRegions:

RNA was isolated from the PUMA1 antibody-secreting hybridoma and wasconverted to 5′ RACE-ready cDNA using the SMARTer RACE 5′/3′ Kit(Clontech, Mountain View, Calif.). Amplification of the heavy chainvariable region was performed using primer CH1(5′-GGCCAGTGGATAGACAGATGG-3′) (SEQ ID NO: 1), while amplification of thelight chain variable region was performed using primer Lk(5′-ACACTCATTCCTGTTGAAGCTCTT-3′) (SEQ ID NO: 2) (primer sequencespublished previously⁶). PCR products of the expected size (roughly 380bp for the heavy chain and 650 bp for the light chain) were ligated intopGEM T-easy (Promega, Madison, Wis.), and multiple isolates sequenced.The predicted light chain variable region was synthesized de-novo(GeneWiz, South Plainfield N.J.) and cloned into pFUSE2-CLIg-hk(Invivogen, San Diego, Calif.). The predicted heavy chain variableregion was synthesized de-novo and cloned into each of the followingvector backbones; pFUSE-CHIg-hG1, pFUSE-CHIg-hG2, pFUSE-CHIg-hG3 andpFUSE-CHIg-hG4 (Invivogen). Using the IgG1-4 backbones as substrates,derivative plasmids encoding each of the known 29 isoallotypes of IgGwere synthesized by a commercial vendor (Genewiz, South Plainfield,N.J., USA).

D. Recombinant Antibody Production:

Recombinant antibodies were produced via transient co-transfection ofthe plasmids encoding the PUMA1 light chain and appropriate PUMA1 heavychain into suspension CHO cells as part of the FreeStyle MAX CHOExpression System (ThermoFisher). Briefly, 24 hr prior to transfection,suspension CHO cells were seeded at a density of 0.5×10⁶ cells/ml.Transfections were performed using a heavy chain to light chain plasmidratio of 2:3, and cultures were grown at 37° C. for 7 days. To harvest,cultures were centrifuged at 4000 RPM for 30 min at 4° C., followed byfiltration of the supernatant through a 0.45 um filter apparatus toremove any remaining cell debris. In some cases, antibodies werepurified from supernatants using rProteinA/G columns (GE Healthcare,Pittsburgh, Pa.), dialyzed, aliquoted and stored at −20° C. Samples fromeach purification were assessed by SDS-PAGE for both purity andconcentration by comparison to a standard curve of purified PUMA1 ofknown concentration.

E. Flow Cytometry:

Flow cytometry consisted of incubating test RBCs with PUMA1 variants,followed by the anti-IgG being evaluated, followed by a detectionreagent. Test RBCs included RBCs from K mice, wild-type mice, andreagent RBCs from humans with the phenotype of (K+k+) or (K−k+). TestRBCs were resuspended in 50 microliters of supernatants of PUMA1isoallotypes; in some cases a 1/10 dilution in phosphate buffered saline(PBS) was used. For purified IgG1-IgG4 PUMA1, the antibodies werediluted in PBS at the indicated concentrations. The tested anti-IgGreagents were used undiluted. Detection reagents consisted of donkeyanti-rabbit conjugated to phycoerythrin at a 1/200 dilution (Affymetrixcat #12-4739-81, Santa Clara, Calif.), goat anti-mouse IgM conjugated toallophycocyanin at a 1/100 dilution (BD Biosciences, cat #550826, SanJose, Calif.), and goat anti-mouse Igs conjugated to allophycocyanin ata 1/100 dilution (SouthernBiotech cat #1020-11s, Birmingham, Ala.). Allanti-IgG subclass antibodies were conjugated to phycoerythrin, werepurchased from SouthernBiotech (catalog numbers 9056-09, 9070-09,9210-09, 9200-09), and were used at a dilution of 1.200. All incubationswere performed for 30 min at room temperature, followed by three washeswith PBS. All flow cytometry was performed on an Accuri 4 colorcytometer (BD biosciences, San Jose Calif.) and all data was analyzedwith Flo-Jo version 10.

F. Solid Phase Testing:

Daily instrument maintenance and quality control were performed asdescribed in the Galileo Echo (Immucor, Norcross, Ga.) Operator Manualprior to testing. The PUMA1 subtypes were tested using combinations ofin-date lots of Capture-R Ready Screen 3 strips (CRRS3), Capture LISSand Capture-R Indicator Cells (CRIND) on 2 Galileo Echo instruments(Immucor). The instrument reads and interprets the test results of theindividual test wells, which in the case of CRRS3 meant that one of thethree test wells contained a K+k+ red cell monolayer and 2 K-k+ red cellmonolayers. The antibody samples were evaluated on 2 instruments using 2lots of CRRS3 strips and 2 lots of CR-IND. Two lots of Capture-Pindicator cells were also used.

G. Tube Antiglobulin Test (AGT):

PUMA1 was tested with Panoscreen I, II & III reagent RBCs (Immucor) by astandard saline tube AGT as described in the direction insert. Briefly,equal volumes of antibody and 2-4% reagent RBCs were incubated for 45minutes at 37° C. After incubation the tubes were washed 3 times with anexcess of PBS. Anti-IgG reagent was added and the tubes centrifuged andthen examined for the presence of agglutination. Gammaclone MurineMonoclonal Anti-IgG (Immucor) was used as Anti-IgG reagents.

H. Gel Testing:

Gel testing was carried out using antibody-screening reagent RBCs (0.8%Surgiscreen RBCs I, II, and III) MTS Anti-IgG Cards, as permanufacturer's instructions (ID-Micro Typing System Gel Test; OrthoClinical Diagnostics, Inc., Raritan, N.J.).

2. Results

A. Generation of a Novel Monoclonal Anti-K Antibody:

RBCs were utilized from previously reported K transgenic mice as animmunogen. Mice with high-titer antisera specific for K transgenic RBCswere identified, spleens were harvested, and fusions were performed withan immortal myeloma line. Through traditional cell cloning methods, anew monoclonal line (PUMA1) was isolated, that secreted an antibody withspecificity for the K antigen. PUMA1 was reactive with RBCs from the Ktransgenic donor mouse used for immunization; no signal was observed onwild-type RBCs compared to secondary antibody alone (FIG. 26A).Similarly, PUMA1 was reactive with human RBCs of the K+k+ phenotype butnot with K-k+ RBCs (FIG. 26B). Characterization of PUMA1 indicates thatit was of the murine IgG2a subclass and expressed a kappa light chain(data not shown).

B. Detection of IgG Subtypes and Isoallotypes by Different Anti-IgGReagents

In order to allow engineering of the PUMA1 antibody, the cDNA for theheavy and light chains were isolated from the PUMA1 hybridoma, and thecoding region for the antibody complementary determining region (CDR)was determined. The heavy chain CDR was cloned, including a Kozaksequence, ATG, and leader sequence, in frame, into expression vectorsfor human IgG1, IgG2, IgG3, and IgG4 (FIG. 26C). Each expression vectorwas used as a starting material to generate additional expressionvectors for the known isoallotypic variants of IgG1-4¹. Likewise, thelight chain CDR was cloned in frame, into an expression vector for humankappa light chain. The plasmid encoding the light chain was thenco-transfected with expression vectors for each of the canonical IgGsubclasses and isoallotypes, and cell culture supernatants whichexpressed PUMA1 IgG variants were collected. Each PUMA1 variant wasincubated with K+k+ RBCs, followed by incubation with either monoclonalor polyclonal anti-IgG, followed by the appropriate detection reagents(see methods). Binding of PUMA1 variants was assessed by flow cytometry.Background staining was determined using K-k+ RBCs that don't expressthe K antigen.

All 29 isoallotypes of human IgG were detected by polyclonal anti-IgG.In contrast, the tested monoclonal anti-IgG bound to 24 out of 29isoallotypes, failing to recognize IgG3-03, IgG3-13, IgG4-01, IgG4-02,and IgG4-03 (see FIG. 27). The lack of detection of these isoallotypicvariants was not due to their lack of expression or inability torecognize the K antigen, as equivalent signal was detected on K+k+ butnot K-k+ RBCs for all 29 isoallotypes by polyclonal anti-IgG.

C. Characterization of Anti-IgG Regents for Sensitivity to Human IgGSubclasses.

To allow standards of known quantity, so as to allow precisedetermination of the sensitivity of anti-IgG, the canonical forms ofIgG1-IgG4 were expressed and purified by affinity chromatography (Allcanonical forms have an isoallotype designation of *01). Proteinelectrophoresis was performed on each purified IgG subclass of PUMA1,both to assess expression and purity (FIG. 26A). The same single bandwas observed at 25 kD for each preparation, consistent with the samekappa light chain of PUMA1. In addition, bands corresponding to each ofthe heavy chains were also observed, at the predicted molecular weightsconsistent with the known size of each of the IgG subclasses. Aspredicted, only the IgG3 heavy chain displayed a higher molecular weightin accordance with its longer hinge region. To test if the purifiedPUMA1 IgG1-IgG4 maintained antigen binding properties and as a furtherconfirmation as to the correct expression, of IgG subtypes, K+k+ humanRBCs were stained with each of the PUMA1 preparations, followed bysecondary antibodies specific for the human IgG subclasses. Each of thePUMA1 IgG subclasses bound to (K+k+) but not (K-k+) RBCs, was reactivewith the secondary antibody specific for the appropriate IgG subclass,and was nonreactive with secondary antibodies of other specificities(FIG. 26B). Together, these findings demonstrate the successfulpurification to homogeneity of the expressed panel of antibodies.Accordingly, determination of protein concentration in thesepreparations reflects the quantity of anti-K IgG, allowing quantitativestandard reagents.

To assess the sensitivity of commercially available anti-IgGpreparations for different human IgG subclasses bound to RBCs, K+k+ RBCswere first incubated with PUMA1 of the different human IgG subclasses,followed by incubation with anti-IgG. Treated RBCs were then stainedwith a fluorescently labeled antibody specific for the species in whichthe anti-IgG was generated (and non-reactive with human IgG). For eachanti-IgG, the same secondary antibody was used to detect binding of theanti-IgG to each human PUMA1 IgG subclass. To assess relativesensitivity, titrations of each PUMA1 IgG subclass were carried out andsamples were analyzed by flow cytometry using mean fluorescent intensity(MFI) to quantify staining (FIGS. 28A, 28B). The polyclonal rabbitanti-IgG reagent produced by Ortho Diagnostics (Ortho anti-IgG) had anincreased sensitivity for IgG3 compared to other IgG subtypes (FIG.28A). In contrast, the mouse monoclonal anti-IgG reagent produced byImmucor-Gamma (Immucor Gamma anti-IgG) had equivalent sensitivity forIgG1, IgG2, and IgG3; however, as observed in the qualitative screeningabove (see FIG. 27), the Immucor Gamma anti-IgG had no detectablereactivity with IgG4 (FIG. 28B).

D. Specificity of Anti-IgG Reagents in Common Platforms Used inImmunohematology Labs

Flow cytometry remains a technique predominantly utilized for researchpurposes. Accordingly, the select forms of PUMA1 IgG1-IgG4 wereevaluated in assay systems and on platforms currently in use in clinicalimmunohematology labs. Five blinded samples were analyzed by theBloodworksNW Immunohematology Reference Laboratory and also by theresearch and development labs at Immucor Inc. The samples consisted ofthe canonical PUMA1 IgG1-IgG4 subtypes or PBS as a negative control. Theblinded samples were evaluated using both solid phase assay systems(Galileo Echo platform, Immucor), by tube testing, or using gel. Overallresults are shown, which were in agreement with flow cytometry results(Table 1). In all cases, systems that utilized the Immucor monoclonal16H8 based reagent, either by solid phase (Capture-R indicator cells) ortube testing with Gammaclone anti-IgG reagent, detected. PUMA1 IgG1,IgG2, and IgG3 (but not IgG4) Capture-P indicator cells are preparedusing a polyclonal rabbit anti-IgG, and detect all 4 PUMA1 IgGsubclasses. In other systems using polyclonal rabbit anti-IgG fromOrtho, all four IgG subclasses of PUMA1 were detected.

Additional gel testing and wet tube testing was carried out on the IgG3isoallotypes that were not detected by the monoclonal anti-IgG usingflow cytometry. Consistent with the flow cytometry results, thecanonical IgG3-01 was detected by Gamma clone reagent in wet tubetesting and in gel; however, neither IgG3-03 nor IgG3-13 was detected ineither platform. Also consistent with flow cytometry, the polyclonalanti-IgG detected IgG3-01 and IgG3-03 both in wet tub testing and ingel. In no case was a positive signal observed in the PBS controlsample. Positive signals were also not detected whenever K-k+ RBCs wereused as screening RBCs (data not shown). Thus, in all cases, standardlyutilized immunohematology methods generated data that was in agreementwith flow cytometry. Sensitivity titrations were not carried out in eachof the clinically used immunohematology platforms.

3. Discussion

The sensitivity and specificity of the anti-IgG component of AHGreagents are essential for immunohematology labs to detect RBCalloantibodies and autoantibodies, most of which are not directagglutinins. However, human IgG is not a monomorphic entity; rather, itconsists of 4 distinct IgG subclasses (IgG1-IgG4), each of which havenatural genetic variation in their constant regions, giving rise to atleast 29 isoallotypes. The subject embodiments are the first assessmentof anti-IgG reactivity to the different human IgG isoallotypes. Thedifficulty in generating test systems of this type have beenacknowledged in past efforts; however, the current use of, for example,recombinant antibodies circumvents the previous barriers. By expressinga panel of antibodies with the same antigen-binding domain, butdifferent IgG constant regions, isoallotype has been isolated as anindependent variable.

Monoclonal anti-IgGs have a number of distinct advantages, including therelative ease of production and consistency of the reagent over time.Moreover, they do not require the ongoing immunization and housing ofanimals to maintain a polyclonal antisera, which can vary frombatch-to-batch. However, the downside to monoclonal reagents can be amore myopic focus on a smaller number of epitopes (or a single epitope)on the target molecule, potentially decreasing the range of recognizedentities. In aspects of the current disclosure, a commonly usedmonoclonal anti-IgG does not recognize 5 of the 29 known isoallotypes ofhuman IgG.

IgG3-03 and IgG3-13 are found at their highest frequencies in a numberof ethnic groups of African origin⁷. IgG3 is typically considered aclinically significant IgG subtype, which is often associated withhemolytic pathology⁸; however, patients with IgG3 and no hemolyticanemia have also been described. The hemolytic potential of differentIgG isoallotypes has not previously been assessed; thus, it is unclearwhere IgG3-03 and/or IgG3-13 fall on the hemolytic spectrum.Juxtaposition of the amino acid sequences indicates that the presence ofa glutamic acid (instead of glutamine) at position 419 is a commoncharacteristic of the IgG3-03, IgG3-13, and each of the IgG4isoallotypes. Thus, the Q to E changes can be responsible for analteration in epitope recognized by the characterized monoclonalanti-IgG. As IgG4 is generally considered to not cause acute hemolyticpathology, it is possible that Q to E change in IgG3-03 and IgG3-13disrupts IgG3 effector function. However, given the potential forhemolysis by IgG3 in general, prudence would dictate an assumption ofhemolytic potential by IgG-03 and/or IgG-13, until proven otherwise.

The non-reactivity of Immucor Gamma anti-IgG with IgG4 is a previouslyknown property of this particular monoclonal antibody (clone 16H8)¹¹,which is listed as a limitation of the antibody in its package insert.The observation that this non-reactivity extends to different IgG4isoallotypes is a novel observation contained herein. Although there areno data on the hemolytic potential of different IgG4 isoallotypes, it isprecisely because IgG4 is typically considered benign that the inabilityof the monoclonal Immucor Gamma anti-IgG (clone 16H8) to bind IgG4 hasbeen acceptable. Indeed, it has been argued that since IgG4 aretypically benign, then non-reactivity to IgG4 is of benefit, since itavoids costly and time-consuming serological workups, which canultimately have a negative impact on patient care through delay in bloodproduct delivery.

While IgG4 is not associated with acute hemolytic events, it is unclearthat IgG4 is entirely benign. Studies by Baldwin et al. convincinglydemonstrated that an anti-JMH, which was IgG4, did indeed fail to causean acute hemolytic transfusion reaction after transfusion of a wholeunit of JMH+ RBCs¹². However, whereas short term ⁵¹Cr studies in thispatient showed a greater than 70% 1 hr recovery, the long term T_(1/2)⁵¹Cr survival was only 12 days. Thus, while not acutely hemolytic, itdoes appear that an anti-JMH IgG4 can substantially decrease thecirculatory life-span of JMH+ RBCs. This can affect not only long-termefficacy for chronically transfused patients (e.g. increase chances ofiron overload due to need of more units over time), but it is alsounclear if ongoing clearance of RBCs by antibody is a benign process.Such sequelae would not cause signs or symptoms that physicians orpatients would experience or report, as one would need to perform RBCsurvival studies to detect the problem. Accordingly, it is of littlevalue to exclude this as a potential problem by the argument that thisreagent has been used for decades without reports of any problems.Finally, it is possible that an IgG4 alloantibody can predict immuneresponse of an IgG1-IgG3 type upon subsequent transfusion, as it hasbeen shown that repeat exposure can alter IgG subtype¹³. This would notbe due to IgG4 secreting cells further switching, but rather due to anew B cell response or IgM+ memory B cells¹⁴. Thus, on balance, it isunclear if an inability to recognize IgG4 is a desirable or undesirableproperty in an anti-IgG regent.

The monoclonal anti-IgG had the same sensitivity for each IgG subtype,which can be an attribute of a monoclonal reagent that recognizes anepitope that is common to the IgG types that it recognizes. In contrast,the rabbit polyclonal reagent tested was more sensitive to IgG3. It isunclear how such differential sensitivity of anti-IgG for different IgGsubtypes can affect serological testing; however, it raises thepossibility of differential detection of alloantibodies in a givenpatient sample by different anti-IgG as a property of the relativelevels of IgG subtypes, which can change over time as a patient'salloimmune response evolves.

It could be argued that there is little likelihood of an adverse patientoutcome due to the lack of reactivity of some anti-IgG (as demonstratedherein). The patient populations to consider include alloimmunizedtransfusion patients, pregnant women with a possibility of HDN, andpatients with autoimmune hemolytic anemia (AIHA). Detailed studies inAIHA patients have demonstrated that a mixture of IgG subclasses is themost common presentation, and has a stronger association with hemolysisthan isolated IgG subclasses^(15,16). In such patients, the monoclonalanti-IgG would still pick up the anti-RBC antibodies, as a mixture ofIgG subclasses is present. However, a significant number of patientshave also been observed who have only a single detectable IgG subclasson their RBCs. In a combined study of both healthy blood donors who hada positive DAT and patients with AIHA, isolated IgG1, IgG2, or IgG3 wereall observed in the context of clinically significant hemolysis; nohemolysis was observed with isolated IgG4¹⁷.

Although infrequent in the Caucasian population (overall about 1%),IgG3-03 and IgG3-13 have up to a 30% frequency in African populations ofcertain distributions⁷. Thus, it is likely that there are some patientswho are homozygous for either IgG3-03 or IgG3-13. In addition, somepatients are likely to be compound heterozygotes for IgG3-03/IgG3-13.Alternatively, even if patients are heterozygous for IgG3-03 or IgG3-13,B cells that make alloantibodies can develop predominantly from clonesthat express the IgG3-03 or IgG3-13 isoallotype. In such patients, inthe event that an anti-RBC antibody response is predominantly of theIgG3 subtype, then they may not be detected by platforms using themonoclonal anti-IgG characterized herein.

In summary, the subject aspects include an approach for assessing thesensitivity and specificity of anti-IgG for different subtypes andisoallotypes of human IgG. Polyclonal anti-IgG had a differentialsensitivity for IgG subtypes, but recognized all 29 known human IgGisoallotypes. In contrast, one monoclonal reagent had blind spots for 5isoallotypes, of the IgG3 or IgG4 subtype. It is unclear what theclinical significance of these blind spots is; however, given thehemolytic potential of IgG3, caution may necessitate reconsideration ofthis reagent.

The overall approach used herein can be utilized to further assesssensitivity and specificity of other anti-IgG reagents, for anydiagnostic platform that has an anti-IgG component. As knowledge of thehuman genome evolves, and new isoallotypes of IgG are identified, thisapproach can be expanded to continue to refine the understanding of thediagnostic specifics of anti-IgG against human immunoglobulins.

TABLE 1 PUMA1 of the indicated human IgG subclasses was analyzed usingtest RBCs of a K+k+ phenotype and using the indicated platforms (seemethods for details). Echo (M00211) Test Results Tube AGT Capture-RCapture-P Gammaclone Gel Sample Indicator Indicator AHG Ortho Testing IDCells Cells Reagent AHG Ortho (PUMA1- Positive Positive 4+ 4+ 3+ IgG1)(PUMA1- Positive Positive 4+ 3+ 3+ IgG2) (PUMA1- Positive Positive 4+ 4+3+ IgG3) (PUMA1- Negative Positive 0  3+ 3+ IgG4) (PUMA1- UN UN 0  3+ 3+IgG3-03) (PUMA1- UN UN 3+ 3+ 3+ IgG3-6) (PUMA1- UN UN 0  3+ 3+ IgG3-13)(PBS) 0 0 0  0  0  Source Clone Polyclonal Clone Polyclonal Polyclonalof anti- 16H8 Rabbit 16H8 Rabbit Rabbit IgG

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed:
 1. A method to determine the specificity andsensitivity of an anti-human globulin antibody, the method comprising:a) binding the anti-human globulin antibody to a panel of humanantibodies, wherein the human antibodies within the panel are ofdifferent Ig subtypes and comprise: a heavy chain comprising:complementarity-determining region (CDR)1 of SEQ ID NO: 114, CDR2 of SEQID NO: 115, and CDR3 of SEQ ID NO: 116; and a light chain comprising:CDR1 of SEQ ID NO: 117, CDR2 of SEQ ID NO: 118, and CDR3 of SEQ ID NO:119; and b) detecting the binding of the anti-human globulin antibody tohuman antibodies of the panel with particular Ig subtypes, thusdetermining the specificity and sensitivity of the anti-human globulinantibody.
 2. The method of claim 1, wherein the human antibodies areIgG.
 3. The method of claim 1, wherein the different Ig subtypescomprise IgG1, IgG2, IgG3, and IgG4.
 4. The method of claim 1, whereinthe different Ig subtypes comprise an isoallotype of IgG1, IgG2, IgG3,or IgG4.
 5. The method of claim 4, wherein the isoallotype is selectedfrom the group consisting of IgG1-01, IgG1-03, IgG1-05, IgG1-07,IgG1-08, IgG1-01v2, and IgG1-04v2.
 6. The method of claim 4, wherein theisoallotype is selected from the group consisting of IgG2-01, IgG2-02,IgG2-04, and IgG2-06.
 7. The method of claim 4, wherein the isoallotypeis selected from the group consisting of IgG3-01, IgG3-03, IgG3-04,IgG3-06, IgG3-08, IgG3-09, IgG3-11, IgG3-12, IgG3-13, IgG3-14, IgG3-15,IgG3-16, IgG3-17, IgG3-18, and IgG3-19.
 8. The method of claim 4,wherein the isoallotype is selected from the group consisting ofIgG4-01, IgG4-02, and IgG4-03.
 9. The method of claim 1, wherein thehuman antibodies are IgA, IgM, IgE, or IgD.
 10. The method of claim 1,wherein the human antibodies comprise heavy chains comprising SEQ IDNOs: 3 or
 5. 11. The method of claim 1, wherein the human antibodiescomprise light chains comprising SEQ ID NOs: 7 or
 9. 12. The method ofclaim 1, wherein the human antibodies bind to an antigen with anaffinity constant (K_(D)) of less than 1×10⁻⁸ M.
 13. The method of claim1, wherein the method is performed using a fluorescence-activated cellsorting (FACS) assay, a gel testing assay, a tube testing assay, or asolid phase testing assay.